Method for Quantitative Study of Airway Functional
Microanatomy Using Micro-Optical Coherence
Tomography
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Citation
Liu, Linbo et al. “Method for Quantitative Study of Airway
Functional Microanatomy Using Micro-Optical Coherence
Tomography.” Ed. Michael Myerburg. PLoS ONE 8.1 (2013):
e54473.
As Published
http://dx.doi.org/10.1371/journal.pone.0054473
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Public Library of Science
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Final published version
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Thu May 26 06:35:41 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/78568
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Method for Quantitative Study of Airway Functional
Microanatomy Using Micro-Optical Coherence
Tomography
Linbo Liu1,2, Kengyeh K. Chu1,2, Grace H. Houser3, Bradford J. Diephuis1,4,5, Yao Li6, Eric J. Wilsterman1,2,
Suresh Shastry6, Gregory Dierksen1,2, Susan E. Birket7, Marina Mazur6, Suzanne Byan-Parker8,
William E. Grizzle8, Eric J. Sorscher6,7, Steven M. Rowe6,7*., Guillermo J. Tearney1,4,9*.
1 Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 2 Department of
Dermatology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 3 Department of Pediatrics, University of Alabama at Birmingham,
Birmingham, Alabama, United States of America, 4 Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, United States of America,
5 Harvard Medical School, Boston, Massachusetts, United States of America, 6 Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham,
Alabama, United States of America, 7 Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 8 Department of
Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 9 Department of Pathology, Massachusetts General Hospital, Boston,
Massachusetts, United States of America
Abstract
We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (mOCT), for
investigating the functional microanatomy of airway epithelia. mOCT captures several key parameters governing the
function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency,
and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling noninvasive study of dynamic phenomena. Additionally, the high resolution of mOCT reveals distinguishable phases of the
ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial
epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard
methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and
quantified.
Citation: Liu L, Chu KK, Houser GH, Diephuis BJ, Li Y, et al. (2013) Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical
Coherence Tomography. PLoS ONE 8(1): e54473. doi:10.1371/journal.pone.0054473
Editor: Michael Myerburg, University of Pittsburgh, United States of America
Received October 11, 2012; Accepted December 11, 2012; Published January 23, 2013
Copyright: ß 2013 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by US National Institute of Health (http://www.nih.gov/; Contracts P30 DK072482 to EJS, R01 HL105487 to SMR, and R01
HL076398 to GJT), Cystic Fibrosis Foundation (http://www.cff.org/; Contracts R464-CF and SORSCH05XX0 to EJS, ROWE10XX0 and CLANCY09Y2 to SMR, and
TEARNE07XX0 to GJT), and Massachusetts General Hospital ECOR Postdoctoral fellowship (http://www.massgeneral.org/; Award 2011A052538). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The following authors declare that they have filed a patent application related to the application of mOCT technology to airways: Linbo
Liu, Kengyeh K. Chu, Grace H. Houser, Bradford J. Diephuis, Eric J. Wilsterman, Gregory Dierksen, Eric J. Sorscher, Steven M. Rowe, and Guillermo J. Tearney. The
application serial number is PCT/US12/52553, entitled ‘‘Method for functional investigation of respiratory airways and other ciliated tissues using mOCT,’’ filed
August 27, 2012. No financial benefit has yet been derived from this application by any author. This does not alter the authors’ adherence to all the PLOS ONE
policies on sharing data and materials.
* E-mail: gtearney@partners.org (GJT); smrowe@uab.edu (SMR)
. These authors contributed equally to this work.
To investigate the pathogenesis, progression, or treatment of
these diseases, a tool to quantitatively characterize the functional
microanatomy of living cells and tissues without disturbing the
mucociliary mechanism is highly desirable. Relevant metrics
include the airway surface liquid (ASL) depth, the thickness of the
thin layer of liquid surrounding the cilia known as the periciliary
liquid (PCL) depth, the ciliary beat frequency (CBF), and the
velocity of mucociliary transport (MCT).
Although individual methods exist for the measurement of ASL,
PCL, CBF, and MCT, each has significant limitations. ASL can be
measured with X–Z scanning confocal microscopy, but requires
transient dyes [5,6] and is not readily performed in vivo. PCL
measurements require osmium tetroxide fixation with perfluorocarbon preservation of the ASL, a technique that is destructive
and cannot be performed on living tissues. Particles and
Introduction
Mucociliary transport and the function of the airway surface is
an area of active study of the human respiratory system. In healthy
airways, a layer of cilia continuously transports airway mucus, a
vital mechanism for defense against particulate contamination and
biological invaders. In many respiratory diseases, however, this
mechanism weakens or fails. Perhaps the best known of these is
cystic fibrosis (CF) airway disease, in which a mutation in the cystic
fibrosis transmembrane conductance regulator (CFTR) impairs
the clearance of mucus from the lungs and airways [1,2,3].
Chronic obstructive pulmonary disease (COPD) also causes
compromised mucus flow [4], as does primary ciliary dyskinesia,
each through distinct mechanisms.
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Study of Mucociliary Clearance Using mOCT
radionuclide labeling can be used to measure the rate of MCT in
vitro and in vivo, respectively [7,8], but the addition of exogenous
material may perturb the mucus transport itself, causing unreliable
measurement [8], and radionuclear studies do not provide cellular
level detail. High frame rate phase contrast microscopy can be
used to measure CBF [9,10,11], but can only be used in transillumination geometries and therefore have limited in vivo potential.
Furthermore, each of these individual techniques involves separate
imaging equipment and contrast mechanisms, a prohibitive
impediment to the comprehensive dynamic study of mucociliary
transport, including the inter-relationships between these parameters, which requires the acquisition of each of these measurements
simultaneously.
Optical coherence tomography (OCT) [12,13] produces crosssectional images based on sample reflectance, which is well suited
for the study of airway microanatomy, since a cross-sectional view
would reveal the thicknesses of the ASL and PCL as well as
capture tissue structure simultaneously. Moreover, since imaging
can be conducted non-invasively and at video rate, functional
parameters can be derived without perturbing the airway surface.
Contrast is derived from the natural reflectance of the various
liquid and cell layers without the need for exogenous dyes, which
enables ASL, PCL, CBF, and MCT measurements simultaneously
from the same set of images. However, though macroscopic
measurements such as mucus transport rate or thick ASL depths
can be made with relatively low resolutions [14], most OCT
systems do not possess sufficient resolution to fully resolve ASL and
PCL layers in normal and CF airway epithelia [15,16,17,18], and
the ciliary layer, a requirement for accurate measurement of CBF.
ASL and PCL thicknesses are critical for maintaining normal
mucociliary clearance, since an ASL depth decrease as small as a
few microns may result in failure of the mucociliary apparatus in
HBE cultures [17], and increases as small as a couple of microns
can contribute to restoration of CF airway epithelial function
[19,20]. Since cilia provide the primary driving force for
mucociliary clearance, the ability to resolve individual cilia and
directly measure their activity including CBF and ciliary beat
pattern is of paramount importance.
The resolution of an OCT system intended for mucociliary
investigation should ideally be smaller than the height difference
between the effective stroke and recovery stroke (,2 mm) [17,21]
in order to investigate the ciliary motion pattern within the PCL
space. The stroke pattern of motile cilia [21], which entails a
complex cycle of recovery, effective forward motion, and rest,
could be revealed with sub-ciliary resolution, which would open to
study an entirely new set of previously inaccessible functional
parameters. A recent publication has indicated that the presence of
ciliary beating can be detected even with an axial resolution of
approximately 3 mm, but a direct quantitative measure of CBF
could not be made, nor was the ciliary stroke pattern imaged [14].
We have developed an OCT system with 1-micron resolution, a
technique we have named micro-OCT (mOCT). Previously
published results from human and swine coronary arteries using
our mOCT system revealed unprecedented subcellular detail in
these tissues [11]. In the present work, we demonstrate mOCT
applied to living airway epithelium, both in cultures and in tissues
ex vivo, including direct and simultaneous measurements of ASL,
PCL, MCT, ciliary stroke pattern, CBF, and glandular function
without exogenous labeling, providing a new tool to interrogate
the functional microanatomy of respiratory epithelia with
unequaled resolution.
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Materials and Methods
Ethics Statement
Use of human cells and tissues was approved by the Institutional
Review Boards at University of Alabama Birmingham (IRB
#X080625002) and Massachusetts General Hospital (IRB
#2008P000178). Primary human bronchial epithelial cells were
derived from lung explants after written informed consent was
obtained from non-CF subjects with confirmed CFTR genetics.
Remnant human tissues following organ explantation were
acquired after written informed consent was obtained from nonCF subjects with confirmed CFTR genetics. The Subcommittee
for Animal Research Care at the Massachusetts General Hospital
(IACUC 2011N000081 and 2010N000242) approved the use of
discarded swine tissue for these studies.
The mOCT system is a spectral-domain OCT implementation
[22,23,24,25] with several key improvements to standard OCT
that yield high resolution in both lateral and axial directions. The
general layout and axial resolution characterization are shown in
Figure 1. A super-continuum source (Fianium SC450) provides the
high-bandwidth, short coherence length light necessary for high
axial resolution (1.3 mm, Fig. 1B). A typical OCT system includes
an interferometer with the reference and sample arms intersecting
at a beamsplitter. The beamsplitter is replaced in mOCT with a 45
degree rod mirror, which apodizes the sample beam by
introducing a circular obscuration in the center to achieve a
balance of good lateral resolution (2 mm) and long depth of focus
(0.2 mm). Custom software is employed to control the galvanometer scanning motors while acquiring spectral data from the line
camera. The system operates with user-configurable line and
frame rates and customizable scan geometry; typical settings are
32 or 40 frames per second, 512 A-lines per frame in a linear scan,
and 0.5 mm by 0.5 mm (X by Z) for a cross-sectional image. The
effective thickness of each cross-section is equal to the mOCT
beam spot size (2 mm).
Primary human bronchial epithelial (HBE) cells were derived
from lung explants according to previously described methods
[26,27]. First or second passage cells following expansion were
seeded on permeable supports for studies. At 80–90% confluency,
cells were seeded onto 1.12 cm2, 12 mm permeable Snap-well
supports (106 cells per filter; Corning Inc., Corning, New York) or
6.5 mm permeable supports (0.56106 cells per filter; Corning Inc.)
that were coated with NIH 3T3 fibroblast conditioned media.
Cells were grown in differentiating media for at least 6–8 weeks
containing DMEM/F12 (Invitrogen, Carlsbad, California), 2%
Ultroser-G (Pall, New York), 2% Fetal Clone II (Hyclone, Logan,
Utah), 2.5 mg/mL insulin (Sigma-Aldrich), 0.25% bovine brain
extract (LONZA), 20 nM hydrocortisone (Sigma-Aldrich),
500 nM Triodothyronine (Sigma-Aldrich), 2.5 mg/mL transferrin
(Invitrogen), 250 nM ethanolamine (Sigma-Aldrich), 1.5 mM
epinephrine (Sigma-Aldrich), 250 nM phosphoethanolamine,
and 10 nM retinoic acid (Sigma-Aldrich) until terminally differentiated.
Normal piglet tracheas were obtained from Exemplar Genetics
(Sioux Center, Iowa). Tissue were dissected from one-day-old
piglets and shipped on wet ice in DMEM. A modified protocol
based on airway tissue handling and preparation methods
developed by Ballard et al. in [28] was employed. Tracheas were
immersed in 80 mL Ringer bicarbonate solution (KRB) baths at
room temperature and slowly warmed to 37uC. After four hours of
pretreatment, the tracheas were removed from the KRB.
Accessible mucus and liquid were aspirated from the airway
lumens and the tracheal ends were cannulated so that the serosal
surface was bathed in KRB [29] without contacting the mucosal
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Study of Mucociliary Clearance Using mOCT
Figure 1. mOCT instrumentation schematic and axial resolution. A. System diagram. RM: reference mirror. OL. objective lens. EC:
environmental chamber. AO: analog output board. G: grating. IMAQ: image acquisition board. L: camera lens. LSC: line scan camera. SMF: single mode
fiber. PC: personal computer. RAID: redundant array of independent disks. CL: Camera Link cable. B. Depth profile of mirror surface, indicating axial
full-width half maximum of 1.3 mm.
doi:10.1371/journal.pone.0054473.g001
previously [33] to acquire images from 4–5 regions of interest in
each well at 100 frames per second. Analysis was performed with
Sisson-Avon Video Analysis (SAVA, Ammons Engineering,
Mount Morris, Michigan). For MCT rate, a 50 mL suspension
of 1 mm diamine polyethylene glycol (PEG) coated fluorescent
beads (Molecular Probes, Eugene, Oregon) was introduced to the
mucus layer. Fluorescence imaging (488 nm excitation, 519 nm
emission) was performed with an inverted microscope (Nikon
Diaphot, Melvin, New York). Images were analyzed using
Metamorph 7.0 software (Molecular Devices, Sunnyvale, California), and transport rate was measured for 10–15 particles per
region.
Excised porcine trachea tissue was also imaged with mOCT,
with ASL, PCL, CBF and MCT measurements acquired with the
same methods as with cultured cells. Additionally, gland ducts
exhibiting mucus extrusion were imaged, and the output flow rate
computed by multiplying velocity (measured in the same manner
as MCT) with the cross-section of the extrusion (measured
geometrically).
Finally, to demonstrate the suitability of mOCT to human tissue
in addition to swine, samples of human trachea tissue were also
imaged, derived from normal donor explant organs not selected
for lung transplantation. Lung, mainstem bronchi, and trachea
were resected en-bloc, transferred on wet ice, and large airways
excised. Airway tissues were then immersed in ice cold DMEM
following resection for transfer, then allowed to equilibrate to
room temperature prior to mOCT imaging.
surface, as previously described [28,30]. Tracheas were allowed to
equilibrate in KRB bubbled with 95% O2 and 5% CO2 at 37uC
and the luminal side exposed to conditioned air at 100% humidity
for 2 hours prior to mOCT imaging [31].
mOCT imaging was performed on HBE cell cultures with
illumination incident on the apical side of the cells. The axis of the
imaging optics is typically placed within 10 degrees of normal to
the cell plane to minimize errors in geometric measurements. ASL
and PCL were measured directly from the thicknesses of the visible
layers in the image with a correction applied for the index of
refraction in the liquid (n = 1.33). ASL and PCL were evaluated at
5 equally distributed regions of the image. CBF and MCT were
determined from a time series of images. CBF was measured by
finding the frequency of peak amplitude in the temporal Fourier
transform of the regions exhibiting oscillatory behavior. Up to 10
regions of ciliary activity per image sequence were assessed for
CBF. MCT was computed by measuring the displacement of 5 to
10 visible inclusions in the mucus through time. All image analysis
was performed with ImageJ and Matlab.
Comparisons to the standard optical methods for ASL, CBF,
and MCT measurements were performed. For all paired
comparisons, imaging was performed 1 mm from the edge of
each well. For ASL depth, the HBE cell surfaces were stained with
Texas Red dye (25 mL at 2 mg/mL in FC-70, administered
2 hours prior to measurements). Transwell membranes were
placed in a sterile glass bottom dish coated with MEM. A confocal
microscope (Carl Zeiss, Oberkochen, Germany) was used to
acquire XZ cross-sectional scans. 4 regions of interest (ROI) were
analyzed for each monolayer [32] and average ASL depth was
measured for 5 equally distributed locations in each ROI. For
CBF, cells were equilibrated at room temperature for 15 minutes,
then evaluated using Hoffman contrast microscopy as described
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Study of Mucociliary Clearance Using mOCT
Results
Imaging respiratory epithelial functional anatomy in live
motion
A representative mOCT image of HBE cells (Fig. 2 A) illustrates
the resolvable features of airway epithelium culture. The epithelial
monolayer and the cilia can be visualized with a resolution
comparable to medium power histology (Fig. 2 B). Mucus and
PCL layer that together comprise the airway surface liquid (ASL)
layer can also be clearly visualized (Fig. 2 A and Movie S1). From
top to bottom, the air has no mOCT signal; the mucus layer
appears heterogeneous with high mOCT signal intensity; the PCL
gel has a low mOCT signal intensity compared with the mucus and
monolayer and includes ciliary structures. The air-liquid interface,
mucus-PCL interface, and apical cell surface are clearly defined so
that ASL and PCL heights can be directly measured with submicrometer resolution. In addition to the different layers, cilia tips
can be readily detected by mOCT, as they are brighter than the
surrounding PCL and mucus. The tips maintain contact with the
deep surface of mucus blanket and lift the mucus nearby by a few
hundred nanometers during the effective stroke (Fig. 2 and Movie
S1), a finding that is consistent with previous observations made
using electron microscopy [21].
Freshly excised full-thickness airway tissue retains functional
mucociliary clearance under physiologic conditions. The mOCT
image of fresh swine tracheal tissue (Fig. 3 A) shows cilia,
epithelium and lamina propia, much as they appeared in histology
(Fig. 3B). ASL and PCL layer can be clearly visualized and directly
measured. A video (Movie S2) demonstrates the activity of the
mucociliary apparatus. Moving mucus and beating cilia are once
again clearly seen and can be readily quantified (Table 1).
In addition to animal tissue, we also imaged human tracheal
tissue acquired from a failed donor lung. mOCT images (Fig. 4 and
Movie S3) of human tissue reveal exactly the same anatomical
features as in HBE cells and animal tissues.
Figure 3. Functional anatomy of excised swine trachea. A. mOCT
image. Yellow bar indicates airway surface liquid (ASL) depth and Red
bar indicates PCL depth. Epithelium (ep) and lamina propria (lp) are also
visible. B. H&E stained histology image illustrating cilia (c), epithelium
(ep) and lamina propria (lp). Scale bar of both images: 10 mm.
doi:10.1371/journal.pone.0054473.g003
Figure 4. mOCT functional anatomy of human trachea. A. A timeaveraged (1 s) mOCT image of human tracheal tissue shows epithelium
(ep), lamina propria (lp), gland duct (gd), mucus (m), cilia (ci) and goblet
cells (gc). Yellow bar indicates airway surface liquid (ASL) depth and Red
bar indicates PCL depth. B. Orthogonal view at the position indicated by
the dashed blue line shows the whole gland duct and the goblet cell in
A. Ciliary beat pattern and possible goblet cell nucleus are clearly seen
in the inset. Scale bars: 20 mm.
doi:10.1371/journal.pone.0054473.g004
Label-free, comprehensive quantification of mucociliary
clearance
confirmed by conventional measures of ASL depth (Fig. 5 A), CBF
(Fig. 5 B), and MCT (Fig. 5 C). Notably, transport rates by particle
tracking were significantly lower than those observed by mOCT,
due to agglomeration of mucus around the fluorescent beads [34].
mOCT MCT rates were not affected by the phenomenon, and
consequently MCT was similar to rates observed in live motion
capture from intact tracheal tissue [35].
mOCT imaging was performed on porcine trachea and the
same techniques previously employed to extract functional
microanatomy data from HBE culture were used in tissue for
the same purpose. The resulting ASL, PCL, CBF, and MCT
numbers are listed in Table 1.
The high resolution and live motion capabilities of mOCT
enable accurate quantification of most of important MCC metrics
without aid of any exogenous contrast agent. Measurements of
ASL, CBF, and MCT from mOCT imaging of HBE culture were
Table 1. mOCT measured parameters from swine trachea ex
vivo.
Figure 2. Representative mOCT image of primary HBE culture.
A. A time-averaged (2 s) mOCT image of fully differentiated primary
human bronchial epithelial cells derived from a normal subject. From
top to bottom, air, mucus layer (mu), PCL layer (pcl; red bar), cilia (green
arrows), and epithelial monolayer (ep) are readily seen. ASL depth is
defined as the distance between the air-mucus interface and the apical
surface of the epithelium (ep). PCL depth is defined by the distance
between the ventral surface of the mucus layer and the apical surface of
the epithelium. B. H&E stained histology of HBE cells. Scale bar: 10 mm.
doi:10.1371/journal.pone.0054473.g002
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Parameter
Value 6 SEM (n = 10)
ASL Depth
9.0160.89 mm
PCL Depth
6.9660.47 mm
CBF
10.5560.12 Hz
MCT Velocity
88.961.8 mm/sec
Number of samples n refers to separate measurements from a single tissue
sample.
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Study of Mucociliary Clearance Using mOCT
Figure 5. Comparison of mOCT and gold standard measurements in HBE cells. All error bars represent SEM. A. ASL depth measured with
mOCT (7.4061.82 mm, n = 5) and confocal microscopy (7.7660.87 mm, n = 6). B. CBF measured with mOCT (9.3260.27 Hz, n = 4) and Hoffman contrast
microscopy (10.1760.56 Hz, n = 4). C. MCT velocity measured with mOCT (24.22614.88 mm/sec, n = 6) and particle-tracking fluorescence microscopy
(1.9160.62 mm/sec, n = 11). Number of measurements n refers to separate wells analyzed.
doi:10.1371/journal.pone.0054473.g005
the relative state of ciliary activity. In mOCT images, cilia tips
appear as high intensity aggregated point scatterers. During the
recovery stroke, the cilia tips appear at lower positions (3–5 mm
from the apical cell surface) than in the effective stroke when they
extend to their full length of ,7 mm (Figs. 7A and 7B, lower
panels). A time-averaged cross-sectional mOCT image shown in
Fig. 7C demonstrates a typical ciliary beat pattern seen in mOCT
images, which is characterized by an arc pattern with a peak 7 mm
above the apical cell surface (yellow arrow) and a bilobular pattern
3–5 mm above the apical cell surface and just below the arc,
indicating recovery strokes (orange arrow). In intact swine trachea,
a similar motion pattern can also be identified (Fig. 7D). An
alternative presentation for beating cilia is M-mode (Fig. 8A),
where the vertical axis is depth and the horizontal axis is time. In
this view, the beating cilia appear as a periodic intensity
modulation. The triphasic pattern [37] of ciliary motion is shown
in Fig. 8 B by decomposing the mOCT ciliary signal in Fig. 8 A
into recovery/rest phase (below 5 mm) and effective phase (above
5 mm). The signal intensity and relative duration of the effective
stroke likely reflect the strength of ciliary motion, potentially
providing information regarding the functional microanatomy of
the relative state of cilia [21].
Functional data was also extracted from videos of active mucus
glands in ex vivo swine tissue. Fig. 6A and Movie S4 shows mucus
extrusion through a gland duct in 2D real-time cross-sectional
images. Additionally, 3D reconstruction of the mOCT image
allows estimation of the gland duct cross-sectional area in the
mucus transport (Fig. 6 B), so that mucus transport rates of luminal
contents can be estimated by multiplying the gland duct crosssectional area with the longitudinal extrusion rates of mucus
estimated from the real-time cross-sectional images. Calculated
average extrusion rate in normal swine under room temperature is
0.095 nL/min (N = 3, 6SEM = 60.006), similar to rates estimated from a previous study [36].
Visualizing ciliary motion in cross-sectional view
The high resolution and live motion capabilities of mOCT also
enable, for the first time to the best of our knowledge, visualization
of ciliary motion in cross-sectional view (Fig. 7 and Movie S5). The
cilia beat cycle can be divided into recovery and effective strokes,
illustrated schematically in the top panels of Figs. 7A and 7B,
respectively, with Fig. 7C showing the full cycle. The recovery
stroke begins with the cilium in fully forward extension (position 0),
then bending and rotating backwards in a clockwise sweep
beneath the mucus. In the effective stroke, the cilium extends
outwards towards the mucus and transcribes an approximately
110u arc in the cross-sectional plane, moving in the direction of
mucus transport [21]. mOCT images provide a means to analyze
Discussion
The high resolution of mOCT enables the straightforward
measurement of key functional parameters from airways: ASL
depth, PCL depth, ciliary beating including CBF, and MCT rate,
as well as extrusion rates of the submucosal airway glands. ASL
and PCL depths are simple geometric measurements that can be
obtained from mOCT images and can be readily discerned due to
the high natural contrast in cells and tissues. To accurately and
sensitively measure PCL, axial resolution must be a fraction of
typical PCL thickness under pathophysiologic condition, which is
approximately 7 mm in normal epithelium and ,3 mm in cell
cultures acquired from cystic fibrosis subjects [17]. The highest
resolution OCT study of airways to date had 3 mm axial resolution
[14], whereas mOCT achieves an axial resolution of 1 mm in tissue.
Additionally, the ability to resolve length scales much smaller than
the PCL itself enables the visualization of objects moving within
the PCL space, such as the beating cilia or particulates within the
mucus. Minute but physiologically significant changes in these
parameters can thus be discerned and sensitively resolved over
Figure 6. Mucus extrusion from single gland duct. A. A
representative frame from a mOCT video of trachea dissected from a
swine shows mucus (yellow arrow) extrusion from a gland duct (gd) in
lamina propria (lp). B. Three-dimensional reconstructed en face view
allows estimation of luminal area of the duct. In swine trachea, mucus
extrusion rate is 0.095 nL/min (N = 3, 6SEM = 60.006) at room
temperature.
doi:10.1371/journal.pone.0054473.g006
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Study of Mucociliary Clearance Using mOCT
time and space, providing the ability to monitor functional
microanatomy. With this resolution, accurate measurements of
CBF can also be obtained directly from a time-series stack of Bmode (cross-sectional) mOCT images by measuring the peak
frequency of oscillatory behavior, as opposed to B-mode speckle
contrast techniques that do not provide quantitative measures of
actual CBF [14]. The speed of mOCT will also allow rapid
scanning of large segments of tissue, a quality that will facilitate in
vivo imaging.
As a result of the high resolution of mOCT, cross-sectional
ciliary stroke pattern can be distinguished in cross-sectional live
imaging for the first time as observed by changes in position (Figs. 7
and 8). The entire beat cycle can be captured if the imaging plane
is oriented such that the cilia tips remain within the 2 mm thickness
of the cross-sectional image through the full beat (Fig. 8).
Alteration in the duration of the effective stroke, the recovery
stroke or the resting state can reflect response to stimulation and
would be expected to confer significant changes on MCC in
addition to CBF itself, in addition to altered ciliary motility. For
example, a number of genes that alter ciliary stroke patterns have
been identified in primary ciliary dyskinesia [38]. Other physiologic stimuli, such as transient and local perturbations of the
airway surface microenvironment induced by pressure
[39,40,41,42] or tonicity [43] are known to alter ciliary beating,
and could be perceived by mOCT for the first time as a means to
analyze the relative state of ciliary activity in living cells and
tissues. Given the structural equivalence of cilia in all mammalian
manifestations, mOCT can also potentially be used to image any
ciliated epithelia, such as ependyma and oviduct, each affected in
significant and common human disease such as hydrocephalus and
infertility, among others.
To validate measurements made by mOCT, comparisons were
made using HBE imaging between mOCT and the gold standard
Figure 7. mOCT images of ciliary motion pattern in HBE culture
and swine trachea. A. (Top panel) 6-stage schematic of ciliary motion
during the recovery stroke; (bottom panel) a mOCT image of fully
differentiated primary HBE cells derived from a normal subject shows
cilia tips (green) 3–5 mm from the apical cell surface, indicating the
recovery stroke. Cilia and mucus are presented in pseudo-colors: green
and purple respectively. B. (top panel) 4-stage schematic of ciliary
motion during the effective stroke; (bottom panel) mOCT signal of the
same cilia after 250 ms that subtend an angle of 114u, delineating an
arc with radius of approximately 7 mm during the effective stroke. C.
(top panel) 10-stage schematic of ciliary motion during the full ciliary
beat cycle; A time-averaged (4 s) image (bottom panel) shows an arc
indicating the effective strokes (yellow arrows) and bilobular pattern of
the recovery stroke (orange arrows). D. A time-averaged (1 s) image of
normal swine trachea shows arcs indicating the effective strokes (yellow
arrows) and bilobular pattern suggesting the recovery stroke (orange
arrows) in the ciliary motion pattern. Scale bars: 10 mm.
doi:10.1371/journal.pone.0054473.g007
Figure 8. Cilia motion pattern in cultured HBE cells. Time-lapsed ciliary motion pattern can be easily identified in the M-mode image (top) of
the active epithelial area shown in Figs. 7 A–C and also Movie S5. The continuity of the sinusoidal pattern in the M-mode image indicates that the
entire beat cycle was captured. Corresponding time-lapse intensity analysis (bottom) reveals triphasic pattern of the ciliary beat cycle: the recovery
stroke (blue line), the effective stroke (orange line) and the rest phase in between the effective stroke and next effective stroke.
doi:10.1371/journal.pone.0054473.g008
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January 2013 | Volume 8 | Issue 1 | e54473
Study of Mucociliary Clearance Using mOCT
techniques previously available for ASL depth, MCT rate, and
CBF measurements. The ASL measurements were in very close
agreement (Figure 5A). The CBFs measured with mOCT and
Hoffman microscopy were also within the margin of error
(Figure 5B), though we noted the Hoffman results were
systematically elevated relative to the mOCT measurements from
the same wells, possibly as a result of a temperature increase
during prolonged microscope illumination during Hoffman
imaging. MCT rate differed significantly between mOCT and
the fluorescence particle tracking method (Figure 5C). However,
the mOCT measurement of 24.2 mm/s is much closer to published
values for bronchial mucus velocity of approximately 40 mm/s
[32,44]. We believe that the introduction of exogenous fluorescent
particles artificially depressed the mucus transport rate, as
evidenced by mucus bundling upon fluorescent imaging, which
further highlights the mOCT advantage of label-free measurement
of MCT.
In our imaging of ex vivo normal porcine trachea (Table 1), we
found functional parameters of similar magnitude to published
data. A previous study of porcine trachea reported a CBF of
12.662.4 Hz and mucus velocity of 4268 mm/s [7]. Measured
PCL depth is also consistent with the typical 7-mm height for
normal airways [17]. ASL depth in tissue is highly dependent on
sample conditions and timing, but the mOCT-derived result
appears reasonable given similar validated measurements from
HBE cells.
Measurement of the output flow rate of mucus glands was
achieved with mOCT and is another novel capability unique to
this technique. Since glandular function is a key constituent of the
function anatomy of the airway surface, is known to be perturbed
in CF airway tissues [45,46] and is responsive to physiologic
stimuli [47], the ability to measure glandular function in situ,
without microdissection, is a significant advance that could reveal
new insights into airway disease pathogenesis and response to
therapeutics.
The availability of a single imaging technology that can unify
the measurement of many functional parameters of the airway
epithelial surface opens the door for many research applications.
Any study of the response of the mucociliary transport apparatus
to a given condition or stimulus can now employ mOCT imaging
to measure these key metrics. For example, the effect of treatments
meant to restore mucus clearance in diseases with mucociliary
impairment can be rapidly measured with mOCT. Imaging of the
ciliary stroke pattern may also facilitate basic science research on
the micro-biomechanics of mucus clearance. Because the technique is rapid and non-invasive, mOCT could also be suitable for
cell-based screening of candidate pharmacologic agents that
modulate the functional airway microanatomy.
mOCT has high potential for translation to in vivo airway
imaging, both in animals and humans. Imaging results from
human tissue (Fig. 4) also illustrate that mOCT can be extended
from culture or animal models to intact human organs. Our ability
to study tissue in situ is limited only by physical access. The
interferometer optics of our present instrument can be replaced by
a fiber probe that can be inserted into an airway lumen, facilitating
endobronchial acquisition guided by fiber optic bronchoscopy, as
previously described for conventional OCT imaging [48]. Such an
advance could provide a crucial step forward in our understanding
of the cellular mechanisms of mucociliary clearance and the
response to experimental therapeutics.
PLOS ONE | www.plosone.org
Conclusion
We have developed mOCT, a high-resolution spectral domain
OCT imaging technique, and established methods and algorithms
to apply mOCT to quantitatively study functional microanatomy
of airways cells and tissue. The high resolution of mOCT allows
many functional parameters to be measured simultaneously and
directly, enabling comprehensive study of the mucociliary
clearance apparatus. Of note, mOCT provides the live visualization of the phases of ciliary strokes, which is not achievable by any
other current method, and readily discerns the PCL depth.
Comparisons with measurements from existing techniques and
known values from the literature have validated mOCT as a
quantitative tool that is well suited for further in vitro and ex vivo
investigation, cell-based functional screening and ultimately,
translation for human use in vivo. Our future work will employ
the imaging system and methods described in this article to
compare CF vs. non-CF phenotypic characteristics and investigate
functional responses to pharmacologic stimulation.
Supporting Information
Representative mOCT movie of primary HBE
culture mucociliary transport. A real-time movie of cultured
HBE cells from a normal donor. See Figure 2 for detailed
explanation of anatomy. Mucus is flowing from right to left above
a layer of beating cilia protruding from the epithelium. From this
image series, MCT rate can be computed by measuring the
displacement of the visible inclusions in the mucus layer through
time, and CBF can be measured by finding the peak frequency of
the temporal Fourier spectrum of the oscillating cilia. ASL and
PCL depth can also be computed from individual frames or from
several averaged frames.
(AVI)
Movie S1
Movie S2 mOCT video of excised porcine tissue with
active mucociliary transport. A thin layer of mucus can be
seen transported from left to right by cilia. Anatomic layers labeled
in representative frame. From top: lumen (L), mucus (mu), cilia
and periciliary layer (PCL), epithelium (ep), and lamina propria
(lp). Scale bar: 10 mm.
(AVI)
Movie S3 mOCT video of human tracheal tissue from
failed donor lung.
(AVI)
Movie S4 mOCT video of mucus extrusion from single
gland duct in swine trachea tissue at room temperature.
(AVI)
Movie S5 mOCT video of ciliary motion. Cilia and mucus
are presented in pseudo-colors: green and purple respectively. The
ciliary pattern is clockwise as the mucus is moving left-to-right.
Scale bar: 10 mm.
(AVI)
Acknowledgments
The authors acknowledge Arianne Fulce, Kathy Sexton, Thurman
Richardson and the Tissue Collection and Banking Facility at UAB for
services related to airway tissue procurement. The authors also
acknowledge Gina Sabbatini and Heather Hathorne for regulatory support
for work with human subjects and the patients who donated their organs
for these experiments. The authors also acknowledge assistance from the
UAB Imaging Core Facility and Professor Mei Wu’s laboratory in
Wellman Center of Photomedicine, MGH. Swine airway tissue was
7
January 2013 | Volume 8 | Issue 1 | e54473
Study of Mucociliary Clearance Using mOCT
obtained from Examplar Genetics Inc. and the MGH Knight Surgical
Laboratory.
Analyzed the data: GJT SMR LL KKC GHH BJD YL EJW SS GD SEB
WEG. Contributed reagents/materials/analysis tools: GJT SMR LL KKC
GD MM SBP WEG EJS. Wrote the paper: GJT SMR LL KKC.
Author Contributions
Conceived and designed the experiments: GJT SMR LL EJS. Performed
the experiments: LL KKC GHH BJD YL EJW SS GD SEB MM SBP.
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